Fetal Alcohol Spectrum Disorders (FASD) affect as many as 9 out of 1,000 pregnancies in the US, and up to 40% of women report consuming some level of alcohol during their pregnancies. Numerous studies have been published characterizing the effect of ethanol on a developing fetus. However, the specific mechanisms have been proven more difficult to establish. The role of the placenta, for example, has been well established in FASD yet there is a lack of definitive biomarkers to define placental phenotypes resulting from ethanol exposure. We propose to study the earliest stages of placental formation, specifically at the level of trophectoderm differentiation, to establish what effect ethanol may have on this critical stage of development. In order to understand how alcohol could affect the motility and establishment of the trophectoderm, we will study the effect of ethanol on the cellular microtubule network. Comprised of 1- and 2-tubulin subunits, microtubule assembly are tightly regulated to ensure proper execution of intracellular organization, protein trafficking, cell migration, and cell division. Acetylation of 1-tubulin at its lysine-40 residue has been shown to affect microtubule assembly dynamics and cell motility. The NAD-dependent sirtuin SIRT2 and the histone deacetylase HDAC6 are responsible for regulating this acetylation. The metabolism of ethanol produces both NADH and acetaldehyde at the cost of NAD+. We propose that ethanol may directly interfere with the ability of SIRT2 and HDAC6 to deacetylate 1-tubulin via the production of acetaldehyde and cellular-depletion of NAD+, thus affecting the overall microtubule network. Using live-imaging, FRAP analysis, in vivo and in vitro mouse pre-implantation models, migration and cell-sorting assays, we will investigate how cell polarity and migration are affected by ethanol in mouse embryonic fibroblasts, mouse embryonic stem cells, and pre-implantation mouse embryos. Furthermore we will establish the effect of ethanol on the microtubule network as well as the mechanism of inhibition of SIRT2/HDAC6 deacetylation. We propose the following specific aims: 1) we will examine how cell polarity and migration are affected by the ethanol-induced disruption of the SIRT2/HDAC6-dependent deacetylation of 1-tubulin; 2) we will Investigate the impact of ethanol on the pre-implantation mouse embryo in regard to cell polarity, migration, and the expression of key developmental genes; 3) we will establish a direct mechanistic link between ethanol, SIRT2, HDAC6, and the deacetylation of 1-tubulin. These experiments will allow us to establish a specific mechanism for the ethanol-induced impairment of implantation and placentation. PUBLIC HEALTH RELEVANCE: Fetal Alcohol Spectrum Disorders affects as many as 9 out of 1,000 pregnancies in the US, and up to 40% of women report consuming some level of alcohol during their pregnancies. The proposed project studies the molecular mechanism for ethanol-induced impairment of fetal growth and will lead to better therapeutic strategies and advisory practices for women who drink while pregnant or while they may become pregnant.